Pharmaceutical combination comprising metformin and dihydroquercetin and its use for the treatment of cancer

ABSTRACT

Provided is a novel medicament capable of reducing the side effects of metformin or a pharmaceutically acceptable salt thereof and useful as an anti-malignant tumor agent, in which metformin or a pharmaceutically acceptable salt thereof and dihydroquercetin or a pharmaceutically acceptable salt thereof are combined.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a medicament wherein metformin or apharmaceutically acceptable salt thereof, and dihydroquercetin or apharmaceutically acceptable salt thereof are combined.

BACKGROUND OF THE INVENTION

Patent document 1 describes a method for the treatment and prevention ofmetabolic disorders and other diseases by administering a pyrone analog,or a derivative thereof, in combination with one or more additionalagents such as, for example, lipid lowering agents or glucose loweringagents. As one of the examples of the pyrone analog, taxifolin isdescribed and, as one of the examples of the glucose lowering agent,metformin is described. However, this document does not disclose anyspecific example of a combination of metformin and taxifolin, and doesnot describe a synergistic effect against malignant tumors or a lacticacidosis-lowering effect of a medicament containing them in combination.

Document List Patent Document

patent document 1: WO 2010/042886

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

Metformin hydrochloride is commercially available as a biguanide oralhypoglycemic agent, and is known to have an action to activateAMP-activated protein kinase (AMPK).

In recent years, it has been reported that a compound having an actionto activate AMPK (AMPK activating agent) such as metformin, phenformin,oligomycin, dinitrophenol, 2-deoxyglucose,5-aminoimidazole-4-carboxyamide ribonucleotide, hydrogen peroxide,sorbitol, calcimycin,4-hydroxy-3-(2′-hydroxybiphenyl-4-yl)-6-oxo-6,7-dihydrothieno[2,3-b]pyridine-5-carbonitrile(A-769662), galegine, troglitazone, phenobarbital, quercetin,resveratrol, berberine and the like shows an anti-malignant tumor effectby a single use thereof or a combined use with existing anticanceragents, and the development thereof as an anti-malignant tumor agent hasbeen considered.

However, of the above, AMPK activating agents such as metformin,phenformin, 5-aminoimidazole-4-carboxyamide ribonucleotide, sorbitol,calcimycin, A-769662, galegine, troglitazone and the like are associatedwith a problem of severe lactic acidosis in the patients who receivedthe administration.

The present invention aims to provide a novel medicament capable ofreducing the side effects of metformin or a pharmaceutically acceptablesalt thereof and useful as an anti-malignant tumor agent.

Metformin is known to have an action to suppress cancer stem cells. Suchmedicament that suppresses cancer stem cells is expected to not onlyprovide an anti-malignant tumor effect, but also enable prevention ofcancer metastasis and prevention of recurrence.

The present invention also aims to provide a novel medicament having anaction to suppress cancer stem cells and useful as an anti-malignanttumor agent.

Means of Solving the Problems

The present inventors have conducted intensive studies in an attempt tosolve the aforementioned problems and found that a combination ofmetformin or a pharmaceutically acceptable salt thereof anddihydroquercetin or a pharmaceutically acceptable salt thereofsuppresses an increase in the blood lactic acid value induced bymetformin or a pharmaceutically acceptable salt thereof. In addition,the present inventors have found that a combination of metformin or apharmaceutically acceptable salt thereof and dihydroquercetin or apharmaceutically acceptable salt thereof remarkably potentiates theanti-malignant tumor action of metformin or a pharmaceuticallyacceptable salt thereof.

The present inventors have also found that dihydroquercetin or apharmaceutically acceptable salt thereof is effective for theprophylaxis or treatment of pancreatic cancer.

The present inventors have found that dihydroquercetin or apharmaceutically acceptable salt thereof is effective for suppressingpancreatic cancer stem cells.

Based on such finding, the present inventors have conducted furtherstudies and completed the present invention.

Accordingly, the present invention provides the following.

[1] A medicament in which metformin or a pharmaceutically acceptablesalt thereof, and dihydroquercetin or a pharmaceutically acceptable saltthereof are combined, wherein metformin or a pharmaceutically acceptablesalt thereof, and dihydroquercetin or a pharmaceutically acceptable saltthereof are contained in a single preparation, or metformin or apharmaceutically acceptable salt thereof, and dihydroquercetin or apharmaceutically acceptable salt thereof are separately formulated intopharmaceutical compositions and used in combination.[2] The medicament of the above-mentioned [1], wherein metformin or apharmaceutically acceptable salt thereof, and dihydroquercetin or apharmaceutically acceptable salt thereof are contained in a singlepreparation.[3] The medicament of the above-mentioned [1], wherein metformin or apharmaceutically acceptable salt thereof, and dihydroquercetin or apharmaceutically acceptable salt thereof are separately formulated intopharmaceutical compositions and used in combination.[4] The medicament of any of the above-mentioned [1] to [3] for theprophylaxis or treatment of a malignant tumor.[5] The medicament of the above-mentioned [4], wherein the malignanttumor is childhood brain tumor selected from the group consisting ofastroglioma, malignant medulloblastoma, germ cell tumor,craniopharyngioma and ependymoma; adult brain tumor selected from thegroup consisting of glioma, glial tumor, meningioma, pituitary adenomaand neurinoma; head and neck cancer selected from the group consistingof maxillary sinus cancer, pharyngeal cancer, laryngeal cancer, mouthcavity cancer, lip cancer, tongue cancer and parotid cancer; thoraciccancer and tumor selected from the group consisting of small cell lungcancer, non-small cell lung cancer, lung adenomatosis and mesothelioma;gastrointestinal cancer and tumor selected from the group consisting ofesophagus cancer, liver cancer, primary liver cancer, gall bladdercancer, bile duct cancer, gastric cancer, colorectal cancer, coloncancer, rectal cancer, anal cancer, pancreatic cancer and pancreaticendocrine tumor; urinary organ cancer and tumor selected from the groupconsisting of penile cancer, renal pelvis-ureteral cancer, renal cellcancer, testis tumor, prostate cancer, urinary bladder cancer, Wilmstumor and urothelial cancer; gynecological cancer and tumor selectedfrom the group consisting of vulvar cancer, cervical cancer, uterinebody cancer, endometrial cancer, uterus sarcoma, chorionic cancer,vagina cancer, breast cancer, ovarian cancer and ovarian germ celltumor; adult and childhood soft tissue sarcoma; bone tumor selected fromthe group consisting of osteosarcoma and Ewing's tumor; cancer and tumorof endocrine tissue selected from the group consisting of adrenal cortexcancer and thyroid cancer; malignant lymphoma and leukemia selected fromthe group consisting of malignant lymphoma, non-Hodgkin's lymphoma,Hodgkin's disease, multiple myeloma, plasma cell tumor, acute myeloidleukemia, acute lymphatic leukemia, adult T-cell leukemia lymphoma,chronic myeloid leukemia and chronic lymphatic leukemia; and skin cancerand tumor selected from the group consisting of chronicmyeloproliferative disease, malignant melanoma, squamous cell cancer,basal cell cancer and mycosis fungoides.[6] The medicament of any of the above-mentioned [1] to [5] for treatinga patient, for whom an anti-malignant tumor drug other than metforminand a pharmaceutically acceptable salt thereof, and dihydroquercetin anda pharmaceutically acceptable salt thereof provides only an insufficienteffect.[7] The medicament of any of the above-mentioned [1] to [5], wherein oneor more kinds of anti-malignant tumor drugs other than metformin and apharmaceutically acceptable salt thereof, and dihydroquercetin and apharmaceutically acceptable salt thereof are combined.[8] The medicament of the above-mentioned [6] or [7], wherein theanti-malignant tumor drug other than metformin and a pharmaceuticallyacceptable salt thereof, and dihydroquercetin and a pharmaceuticallyacceptable salt thereof is a molecule target drug, alkylating agent,metabolic antagonist, plant alkaloid, anticancer antibiotic, hormoneagent or immunotherapeutic agent.[9] A commercial package comprising the medicament of any of theabove-mentioned [1] to [8] and a written matter associated therewith,the written matter stating that the medicament can or should be used forthe prophylaxis or treatment of a malignant tumor.[10] An agent for reducing the side effect of metformin or apharmaceutically acceptable salt thereof, which comprisesdihydroquercetin or a pharmaceutically acceptable salt thereof as anactive ingredient.[11] An enhancer of an anti-malignant tumor action of metformin or apharmaceutically acceptable salt thereof, which comprisesdihydroquercetin or a pharmaceutically acceptable salt thereof as anactive ingredient.[12] An agent for reducing the side effect of metformin or apharmaceutically acceptable salt thereof comprising dihydroquercetin ora pharmaceutically acceptable salt thereof as an active ingredient,which is used in combination with metformin or a pharmaceuticallyacceptable salt thereof.[13] An enhancer of an anti-malignant tumor action comprisingdihydroquercetin or a pharmaceutically acceptable salt thereof as anactive ingredient, which is used in combination with metformin or apharmaceutically acceptable salt thereof.[14] Use of dihydroquercetin or a pharmaceutically acceptable saltthereof in combination with metformin or a pharmaceutically acceptablesalt thereof in the production of a medicament for the prophylaxis ortreatment of a malignant tumor.[15] A method for the prophylaxis or treatment of a malignant tumor,comprising administering metformin or a pharmaceutically acceptable saltthereof, and dihydroquercetin or a pharmaceutically acceptable saltthereof to a subject in need thereof, wherein metformin or apharmaceutically acceptable salt thereof, and dihydroquercetin or apharmaceutically acceptable salt thereof are administered as a singlepreparation or separate preparations which are administeredsimultaneously or at different time points.[16] A medicament for the prophylaxis or treatment of pancreatic cancer,comprising dihydroquercetin or a pharmaceutically acceptable saltthereof as an active ingredient.[17] A method for the prophylaxis or treatment of pancreatic cancer,comprising administering dihydroquercetin or a pharmaceuticallyacceptable salt thereof to a subject in need thereof.[18] Use of dihydroquercetin or a pharmaceutically acceptable saltthereof as a medicament for the prophylaxis or treatment of pancreaticcancer.[19] Dihydroquercetin or a pharmaceutically acceptable salt thereof foruse in the prophylaxis or treatment of pancreatic cancer.[20] Use of dihydroquercetin or a pharmaceutically acceptable saltthereof in the production of a medicament for the prophylaxis ortreatment of pancreatic cancer.[21] A pancreatic cancer stem cell inhibitor comprising dihydroquercetinor a pharmaceutically acceptable salt thereof as an active ingredient.[22] A method of suppressing a pancreatic cancer stem cell, comprisingadministering dihydroquercetin or a pharmaceutically acceptable saltthereof to a subject in need thereof.[23] Use of dihydroquercetin or a pharmaceutically acceptable saltthereof as a pancreatic cancer stem cell inhibitor.[24] Dihydroquercetin or a pharmaceutically acceptable salt thereof foruse in suppressing a pancreatic cancer stem cell.[25] Use of dihydroquercetin or a pharmaceutically acceptable saltthereof in producing a pancreatic cancer stem cell inhibitor.[26] A medicament comprising metformin or a pharmaceutically acceptablesalt thereof for use in combination with dihydroquercetin or apharmaceutically acceptable salt thereof.[27] A medicament comprising dihydroquercetin or a pharmaceuticallyacceptable salt thereof for use in combination with metformin or apharmaceutically acceptable salt thereof.

Effect of the Invention

The medicament of the present invention containing metformin or apharmaceutically acceptable salt thereof and dihydroquercetin or apharmaceutically acceptable salt thereof in combination has asynergistic anti-malignant tumor effect and is useful as ananti-malignant tumor drug.

The medicament of the present invention is a safe medicament thatreduces lactic acidosis, since dihydroquercetin or a pharmaceuticallyacceptable salt thereof suppresses an increase in the blood lactic acidvalue induced by metformin or a pharmaceutically acceptable saltthereof.

Since the medicament of the present invention has a synergisticanti-malignant tumor effect, it enables low dosing, and further, isexpected to reduce side effects.

The medicament of the present invention containing dihydroquercetin or apharmaceutically acceptable salt thereof as an active ingredient isuseful as a prophylactic or therapeutic drug for pancreatic cancer.

Gemcitabine which is a drug of first alternative for the treatment ofpancreatic cancer permits growth of pancreatic cancer stem cells, asshown in the results of the below-mentioned Example 12.

The medicament of the present invention having a synergistic pancreaticcancer stem cell suppressive effect is particularly useful as aprophylactic or therapeutic drug for pancreatic cancer. In addition, thecombination of the medicament of the present invention and gemcitabineis expected to improve clinical problems of an increase of cancer stemcells and an increase of recurrence risk in the gemcitabine treatment,and is particularly useful as a prophylactic or therapeutic drug forpancreatic cancer.

The medicament of the present invention, containing dihydroquercetin ora pharmaceutically acceptable salt thereof as an active ingredient,shows a pancreatic cancer stem cell suppressive effect. Therefore, it isuseful as a prophylactic or therapeutic drug for pancreatic cancer, andfurther expected to prevent metastasis or recurrence of pancreaticcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of Example 1.

FIG. 2 shows the results of Example 2.

FIG. 3 shows the results of Example 3.

FIG. 4 shows the results of racemate of dihydroquercetin((2R,3R)-dihydroquercetin and (2S,3S)-dihydroquercetin) in Example 10.

FIG. 5 shows the results of optically-active dihydroquercetin((2R,3R)-dihydroquercetin) in Example 10.

FIG. 6 shows the results of Example 11.

FIG. 7 shows the results of Example 12. In the Figure, Met meansmetformin, DHQ means dihydroquercetin, and Gem means gemcitabine.

DESCRIPTION OF EMBODIMENTS (I) Combined Use of Metformin or aPharmaceutically Acceptable Salt Thereof and Dihydroquercetin or aPharmaceutically Acceptable Salt Thereof

Examples of the pharmaceutically acceptable salt of metformin includesalt with inorganic acid, salt with organic acid, salt with acidic aminoacid and the like.

Examples of the salt with inorganic acid include a salt withhydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid and the like. Examples of the salt with organic acidinclude a salt with formic acid, acetic acid, trifluoroacetic acid,fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid,succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and the like. Examples of the salt with acidicamino acid include a salt with aspartic acid, glutamic acid and thelike. Of these, hydrochloride is preferable.

Dihydroquercetin is a compound wherein the double bond between the2-position and the 3-position of the pyran ring of quercetin has beenreduced, and is represented by the following formula:

Dihydroquercetin contains 4 stereoisomers based 2 asymmetric carbonatoms (2-position and 3-position of chromane ring)((2R,3R)-dihydroquercetin, (2S,3S)-dihydroquercetin,(2R,3S)-dihydroquercetin, (2S,3R)-dihydroquercetin). Whiledihydroquercetin in the present invention may be a combination of one ormore kinds of these isomers or a racemate of these (e.g., mixture of(2R,3R)-dihydroquercetin and (2S,3S)-dihydroquercetin), the racemate of(2R,3R)-dihydroquercetin and (2S,3S)-dihydroquercetin, and(2R,3R)-dihydroquercetin, are preferable, and (2R,3R)-dihydroquercetinis particularly preferable.

Of the isomers, it is known that (2R,3R)-dihydroquercetin represented bythe following formula:

is contained in Larix sibirica, and is also called taxifolin. In thepresent invention, naturally-occurring substances such as taxifolinextracted by a known method and the like may also be used, andcommercially available products can also be used.

Examples of the pharmaceutically acceptable salt of dihydroquercetininclude salt with inorganic base, salt with organic base, salt withbasic amino acid and the like.

Examples of the salt with inorganic base include a salt with alkalimetal such as sodium, potassium and the like, alkaline earth metal suchas calcium, magnesium and the like, and aluminum, ammonium and the like.Examples of the salt with organic base include a salt withtrimethylamine, triethylamine, pyridine, picoline, ethanolamine,diethanolamine, triethanolamine, dicyclohexylamine,N,N-dibenzylethylenediamine and the like. Examples of the salt withbasic amino acid include a salt with arginine, lysine, ornithine and thelike. Examples of a preferable salt include alkali metal salt andalkaline earth metal salt.

The metformin and a pharmaceutically acceptable salt thereof, anddihydroquercetin and a pharmaceutically acceptable salt thereof usablein the present invention also encompass the same isotope labeledcompounds wherein one or more atoms are replaced by one or more atomshaving particular atom mass or mass number. Examples of the isotope thatcan be incorporated into these compounds include hydrogen, carbon,nitrogen, oxygen, sulfur, fluorine, and chlorine isotope such as ²H, ³H,¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³³S, ³⁴S, ³⁶S, ⁸F, ³⁶Cl and the like. Aparticular isotope-labeled compound containing the above-mentionedisotope and/or other isotope of other atom, for example, a compoundincorporating a radioactive isotope such as ³H, ¹⁴C and the like isuseful for drug tissue distribution assay and/or substrate tissuedistribution assay. A tritiated (i.e., ³H) isotope and carbon-14 (i.e.,¹⁴C) isotope are particularly preferred in view of the easiness ofpreparation and detectability. Furthermore, substitution with a heavierisotope such as deuterium (i.e., ²H) and the like is expected to improvemetabolic stability, for example, by affording particular advantages inthe treatment due to an increase in the in vivo half-life or a decreasein the necessary dose.

In the medicament of the present invention, metformin or apharmaceutically acceptable salt thereof and dihydroquercetin or apharmaceutically acceptable salt thereof are used in combination. In themedicament of the present invention, metformin or a pharmaceuticallyacceptable salt thereof, and dihydroquercetin or a pharmaceuticallyacceptable salt thereof may be simultaneously formulated or may becontained in the same preparation. Alternatively, metformin or apharmaceutically acceptable salt thereof, and dihydroquercetin or apharmaceutically acceptable salt thereof may be separately formulated,and may be administered to a single subject simultaneously or atdifferent time points by the same pathway or different pathways. Thatis, the medicament of the present invention includes a medicamentcontaining metformin or a pharmaceutically acceptable salt thereof, anddihydroquercetin or a pharmaceutically acceptable salt thereof in asingle preparation, and a medicament using a pharmaceutical compositioncomprising metformin or a pharmaceutically acceptable salt thereof and apharmaceutical composition comprising dihydroquercetin or apharmaceutically acceptable salt thereof, which are formulatedseparately, in combination.

The medicament of the present invention can be formulated into, forexample, a pharmaceutical composition such as a tablet (includingsugar-coated tablet, film-coated tablet), a powder, a granule, a capsule(including soft capsule), a liquid, an injection, a suppository, asustained-release preparation (e.g., sustained-release microcapsule), oran immediate-release preparation by, for example, mixing metformin or apharmaceutically acceptable salt thereof, and/or dihydroquercetin or apharmaceutically acceptable salt thereof with a pharmacologicallyacceptable carrier according to a known method, and safely administeredorally or parenterally (e.g., topical, rectal, intravenousadministration etc.). The injection can be used for intravenous,intramuscular, subcutaneous or intraorgan administration, or can bedirectly administered to the lesion.

Examples of the pharmacologically acceptable carrier that can be usedfor the production of the medicament of the present invention includevarious organic or inorganic carrier substances conventionally used,such as excipient, lubricant, binder and disintegrant for solidpreparations, solvent, solubilizing agents, suspending agent, isotonicagent, buffering agent and soothing agent for liquid preparations andthe like. Furthermore, where necessary, appropriate amounts of generaladditives such as preservative, antioxidant, colorant, sweetening agent,adsorbent, wetting agent and the like can also be used.

Examples of the excipient include lactose, sucrose, D-mannitol, starch,cornstarch, crystalline cellulose, light anhydrous silicic acid and thelike.

Examples of the lubricant include magnesium stearate, calcium stearate,talc, colloidal silica and the like.

Examples of the binder include crystalline cellulose, sucrose,D-mannitol, dextrin, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, gelatin,methylcellulose, carboxymethylcellulose sodium and the like.

Examples of the disintegrant include starch, carboxymethylcellulose,carboxymethylcellulose calcium, sodium carboxymethyl starch,L-hydroxypropylcellulose and the like.

Examples of the solvent include water for injection, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, olive oil and the like.

Examples of the solubilizing agent include polyethylene glycol,propylene glycol, D-mannitol, benzyl benzoate, ethanol,trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodiumcitrate and the like.

Examples of the suspending agent include surfactants such asstearyltriethanolamine, sodium lauryl sulfate, lauryl aminopropionicacid, lecithin, benzalkonium chloride, benzethonium chloride, glycerolmonostearate and the like; hydrophilic polymers such as polyvinylalcohol, polyvinylpyrrolidone, carboxymethylcellulose sodium,methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose and the like; and the like.

Examples of the isotonic agent include glucose, D-sorbitol, sodiumchloride, glycerol, D-mannitol and the like.

Examples of the buffering agent include buffers such as phosphate,acetate, carbonate, citrate and the like; and the like.

Examples of the soothing agent include benzyl alcohol and the like.

Examples of the preservative include paraoxybenzoates, chlorobutanol,benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid andthe like.

Examples of the antioxidant include sulfite, ascorbic acid, α-tocopheroland the like.

The contents of metformin or a pharmaceutically acceptable salt thereof,and dihydroquercetin or a pharmaceutically acceptable salt thereof inthe medicament of the present invention can be appropriately selectedaccording to the form of the preparation and the like.

For example, the content of metformin in the medicament of the presentinvention containing metformin and dihydroquercetin in a singlepreparation is generally about 0.01 to about 99.99 wt %, preferablyabout 0.1 to about 50 wt %, relative to the whole preparation, and thecontent of dihydroquercetin is generally about 0.01 to about 99.99 wt %,preferably about 0.1 to about 50 wt %, relative to the wholepreparation.

The content ratio of metformin and dihydroquercetin in the medicament ofthe present invention is about 0.0005-300 parts by weight, preferablyabout 0.5-300 parts by weight, more preferably about 0.5-100 parts byweight, still more preferably about 2.5-50 parts by weight, particularlypreferably about 2.5-10 parts by weight, of metformin per 1 part byweight of dihydroquercetin.

In addition, the content ratio of metformin and dihydroquercetin in themedicament of the present invention is about 1-600 mol, preferably about1-200 mol, more preferably about 5-100 mol, still more preferably about5-20 mol, of metformin per 1 mol of dihydroquercetin.

While the content of the additive such as carrier and the like in themedicament of the present invention varies depending on the form of thepreparation, it is generally about 1 to about 99.99 wt %, preferablyabout 10 to about 90 wt %, relative to the whole preparation.

When metformin and dihydroquercetin are separately formulated, thecontent of metformin in the preparation containing metformin isgenerally about 0.01 to about 100 wt %, preferably about 0.1 to about 90wt %, relative to the whole preparation, and the content ofdihydroquercetin in the preparation containing dihydroquercetin isgenerally about 0.01 to about 100 wt %, preferably about 0.1 to about 90wt %, relative to the whole preparation. The content of an additive suchas carrier and the like is as mentioned above.

The use rate of metformin and dihydroquercetin when they are separatelyformulated is generally about 0.0005-300 parts by weight, preferablyabout 0.5-300 parts by weight, more preferably about 0.5-100 parts byweight, still more preferably about 2.5-50 parts by weight, particularlypreferably about 2.5-10 parts by weight, of metformin per 1 part byweight of dihydroquercetin.

In addition, the use rate of metformin and dihydroquercetin when theyare separately formulated is about 0.001-600 mol, preferably about 1-600mol, more preferably about 1-200 mol, still more preferably about 1-100mol, particularly preferably about 5-20 mol, of metformin per 1 mol ofdihydroquercetin.

When a pharmaceutically acceptable salt of metformin and/or apharmaceutically acceptable salt of dihydroquercetin are/is used, thecontents thereof in the preparation and the content ratio thereof arewithin the same range as that indicated above for metformin anddihydroquercetin.

These preparations can be produced by a known method generally used inthe preparation steps.

In the case of injection, for example, injections (e.g., Captisolpreparation) can be produced by producing an aqueous injection ofmetformin or a pharmaceutically acceptable salt thereof, and/ordihydroquercetin or a pharmaceutically acceptable salt thereof togetherwith dispersing agents (e.g., Tween 80 (manufactured by Atlas Powder,USA), HCO 60 (manufactured by Nikko Chemicals), polyethylene glycol,carboxymethylcellulose, sodium alginate, hydroxypropylmethylcellulose,dextrin etc.), stabilizers (e.g., ascorbic acid, sodium pyrrosulfiteetc.), surfactants (e.g., polysorbate 80, macrogol etc.), solubilizers(e.g., glycerol, ethanol, Captisol (trade name,sulfobutylether-R-cyclodextrin sodium salt) etc.), buffering agents(e.g., citric acid, phosphoric acid and alkali metal salt thereof,citric acid and alkali metal salt thereof etc.), isotonic agents (e.g.,sodium chloride, potassium chloride, mannitol, sorbitol, glucose etc.),pH adjusters (e.g., hydrochloric acid, sodium hydroxide etc.),preservatives (e.g., ethyl parahydroxybenzoate, benzoic acid, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, benzyl alcohol etc.),dissolving agents (e.g., concentrated glycerol, meglumine etc.),solubilizing agents (e.g., propylene glycol, sucrose etc.), soothingagents (e.g., glucose, benzyl alcohol etc.) and the like, or an oilyinjection by dissolving, suspending or emulsifying them in vegetable oilsuch as olive oil, sesame oil, cottonseed oil, corn oil and the like, ora solubilizing agent such as propylene glycol and the like.

In the case of oral preparation (tablet), for example, an oralpreparation can be produced by mixing metformin or a pharmaceuticallyacceptable salt thereof, and/or dihydroquercetin or a pharmaceuticallyacceptable salt thereof and excipients (e.g., lactose, sucrose, starch,cornstarch etc.), disintegrants (e.g., starch, calcium carbonate etc.),binders (e.g., starch, gum arabic, carboxymethylcellulose,polyvinylpyrrolidone, hydroxypropylcellulose, gelatin etc.), lubricants(e.g., talc, magnesium stearate, polyethylene glycol 6000 etc.) and thelike, compression molding the mixture, then, where necessary, coatingsame by a known method for masking of taste, enteric coating orsustainability.

Examples of the coating agent used here includehydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose,hydroxypropylcellulose, polyoxyethyleneglycol, Tween 80, pluronic F68,cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate,hydroxymethylcellulose acetate succinate, Eudragit (manufactured byRohm, Germany, methacrylic acid-acrylic acid copolymer) and dye (e.g.,ferric oxide red, titanium dioxide etc.) and the like. Examples of thesugar coating include sucrose, talc, gum arabic, dye (e.g., ferric oxidered, titanium dioxide etc.), polishing agent (e.g., beeswax etc.) andthe like.

In the case of suppository, for example, metformin or a pharmaceuticallyacceptable salt thereof, and/or dihydroquercetin or a pharmaceuticallyacceptable salt thereof can be formulated into an oily or aqueous solid,semisolid or liquid suppository by using substrates such as oily bases(e.g., glycerides of higher fatty acid [e.g., cacao butter, witepsols(manufactured by Dynamitnovel Ltd., Germany) etc.], medium-chain fattyacids [e.g., miglyols (manufactured by Dynamitnovel Ltd., Germany)etc.], vegetable oils (e.g., sesame oil, soybean oil, cottonseed oiletc.) etc.), aqueous bases (e.g., polyethylene glycols, propylene glycoletc.), aqueous gel bases (e.g., natural gums, cellulose derivative,vinyl polymer, acrylic acid polymer etc.) and the like according to aknown method.

The medicament of the present invention shows reduced side effects andcan be safely administered to human and animals (e.g., mouse, rat,rabbit, dog, cat, bovine, horse, swine, monkey etc.).

The dose of the medicament of the present invention can be appropriatelyselected according to the use, age and sex of the patient, level of thedisease and the like. The dose of the medicament of the presentinvention for an adult (body weight 60 kg) per day is generally about250-3000 mg, preferably 500-2250 mg, of metformin or a pharmaceuticallyacceptable salt thereof, and generally about 40-1200 mg, preferably50-900 mg, of dihydroquercetin or a pharmaceutically acceptable saltthereof. The dose is administered in one to several portions per day.

The medicament of the present invention is useful as a prophylactic ortherapeutic agent for a malignant tumor.

Examples of the malignant tumor include childhood brain tumor selectedfrom the group consisting of astroglioma, malignant medulloblastoma,germ cell tumor, craniopharyngioma and ependymoma; adult brain tumorselected from the group consisting of glioma, glial tumor, meningioma,pituitary adenoma and neurinoma; head and neck cancer selected from thegroup consisting of maxillary sinus cancer, pharyngeal cancer (e.g.,nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer),laryngeal cancer, mouth cavity cancer, lip cancer, tongue cancer andparotid cancer; thoracic cancer and tumor selected from the groupconsisting of small cell lung cancer, non-small cell lung cancer, lungadenomatosis and mesothelioma; gastrointestinal-1 cancer and tumorselected from the group consisting of esophagus cancer, liver cancer,primary liver cancer, gall bladder cancer, bile duct cancer, gastriccancer, colorectal cancer, colon cancer, rectal cancer, anal cancer,pancreatic cancer and pancreatic endocrine tumor; urinary organ cancerand tumor selected from the group consisting of penile cancer, renalpelvis-ureteral cancer, renal cell cancer, testis tumor (also calledorchis tumor), prostate cancer, urinary bladder cancer, Wilms tumor andurothelial cancer; gynecological cancer and tumor selected from thegroup consisting of vulvar cancer, cervical cancer, uterine body cancer,endometrial cancer, uterus sarcoma, chorionic cancer, vagina cancer,breast cancer, ovarian cancer and ovarian germ cell tumor; adult andchildhood soft tissue sarcoma; bone tumor selected from the groupconsisting of osteosarcoma and Ewing's tumor; cancer and tumor ofendocrine tissue selected from the group consisting of adrenal cortexcancer and thyroid cancer; malignant lymphoma and leukemia selected fromthe group consisting of malignant lymphoma, non-Hodgkin's lymphoma,Hodgkin's disease, multiple myeloma, plasma cell tumor, acute myeloidleukemia, acute lymphatic leukemia, adult T-cell leukemia lymphoma,chronic myeloid leukemia and chronic lymphatic leukemia; and skin cancerand tumor selected from the group consisting of chronicmyeloproliferative disease, malignant melanoma, squamous cell cancer,basal cell cancer and mycosis fungoides.

Since the medicament of the present invention has a particularlyremarkable anti-malignant tumor effect, it is expected to show ananti-malignant tumor effect for a patient for whom an anti-malignanttumor drug other than metformin and a pharmaceutically acceptable saltthereof, and dihydroquercetin and a pharmaceutically acceptable saltthereof provides only an insufficient effect.

The medicament of the present invention may be further combined with oneor more kinds of anti-malignant tumor drugs other than metformin and apharmaceutically acceptable salt thereof, and dihydroquercetin and apharmaceutically acceptable salt thereof.

Examples of the anti-malignant tumor drug other than metformin and apharmaceutically acceptable salt thereof, and dihydroquercetin and apharmaceutically acceptable salt thereof include a molecule target drug,an alkylating agent, a metabolic antagonist, a plant alkaloid, ananticancer antibiotic, a hormone agent, an immunotherapeutic agent andthe like.

Examples of the aforementioned “molecule target drug” include imatinib,gefitinib, erlotinib, sunitinib, sorafenib, brivanib, tivantinib,linifanib, bortezomib, nilotinib, dasatinib, lestaurtinib, lapatinib,thalidomide, lenalidomide, sirolimus, everolimus, temsirolimus,vorinostat, tretinoin, tamibarotene, rituximab, bevacizumab,ramucirumab, panitumumab, cetuximab, trastuzumab, alemtuzumab,gemtuzumab, ozogamicin, ibritumomab tiuxetan, azacitidine, decitabine,zoledronic acid, arsenic trioxide, oblimersen and the like.

Examples of the aforementioned “alkylating agent” includemechlorethamine; cyclophosphamide; ifosfamide; carmustine; busulfan;temozolomide; procarbazine; lomustine; dacarbazine; bendamustine;melphalan; nimustine; ranimustine; chlorambucil; fotemustine; platinumpreparations such as oxaliplatin, cisplatin, carboplatin and the like;and the like.

Examples of the aforementioned “metabolic antagonist” includegemcitabine, methotrexate, capecitabine, cytarabine, fludarabine,cladribine, enocitabine, carmofur, tegafur, tegafur⋅uracil,tegafur⋅gimeracil⋅oteracil potassium, 5-fluorouracil, doxifluridine,nelarabine, hydroxycarbamide, pentostatin, mercaptopurine, leucovorin,pemetrexed and the like.

Examples of the aforementioned “plant alkaloid” include topoisomeraseinhibitors such as irinotecan, topotecan, nogitecan, etoposide,sobuzoxane and the like; mitotic inhibitors such as paclitaxel, Abraxane(trade name), docetaxel, ixabepilone, vinblastine, vindesine,vincristine, vinorelbine, eribulin and the like; and the like.

Examples of the aforementioned “anticancer antibiotic” includedoxorubicin, mitomycin C, mitoxantrone, epirubicin, idarubicin,daunorubicin, aclarubicin, amrubicin, pirarubicin, actinomycin D,bleomycin, peplomycin, cyclosporin and the like.

Examples of the aforementioned “hormone agent” include tamoxifen,anastrozole, letrozole, exemestane, fulvestrant, flutamide,bicalutamide, estramustine, chlormadinone, toremifene, goserelin,prednisone, leuprorelin, abiraterone, dexamethasone and the like.

Examples of the aforementioned “immunotherapeutic agent” include GM-CSF,interferon alpha 2b, interleukin 2, filgrastim, epoetin alfa and thelike.

The present invention also relates to a commercial package comprisingthe above-mentioned “medicament in which metformin or a pharmaceuticallyacceptable salt thereof, and dihydroquercetin or a pharmaceuticallyacceptable salt thereof are combined” of the present invention, and awritten matter associated therewith, the written matter stating that themedicament can or should be used for the prophylaxis or treatment of amalignant tumor.

The present invention further relates to an agent for reducing the sideeffect (particularly lactic acidosis) of metformin or a pharmaceuticallyacceptable salt thereof, which comprises dihydroquercetin or apharmaceutically acceptable salt thereof as an active ingredient, and anenhancer of an anti-malignant tumor action of metformin or apharmaceutically acceptable salt thereof, which comprisesdihydroquercetin or a pharmaceutically acceptable salt thereof as anactive ingredient. The present invention also relates to an agent forreducing the side effect (particularly lactic acidosis) of metformin ora pharmaceutically acceptable salt thereof comprising dihydroquercetinor a pharmaceutically acceptable salt thereof as an active ingredient,which is used in combination with metformin or a pharmaceuticallyacceptable salt thereof, and an enhancer of an anti-malignant tumoraction comprising dihydroquercetin or a pharmaceutically acceptable saltthereof as an active ingredient, which is used in combination withmetformin or a pharmaceutically acceptable salt thereof.

In the side effects reducing agent and the anti-malignant tumor actionenhancer, examples of “metformin”, “metformin or a pharmaceuticallyacceptable salt thereof”, “dihydroquercetin or a pharmaceuticallyacceptable salt thereof”, administration form, dose, subject ofadministration, target disease and the like are similar to thosedescribed for the above-mentioned medicament of the present invention.

(II) Medicament Containing Dihydroquercetin or a PharmaceuticallyAcceptable Salt Thereof as an Active Ingredient

The present invention relates to a medicament for the prophylaxis ortreatment of pancreatic cancer, which contains dihydroquercetin or apharmaceutically acceptable salt thereof as an active ingredient, and apancreatic cancer stem cell inhibitor comprising dihydroquercetin or apharmaceutically acceptable salt thereof as an active ingredient.

Examples of the “dihydroquercetin or a pharmaceutically acceptable saltthereof”, administration form, subject of administration and the likeare similar to those described for the above-mentioned (I).

The medicament and the inhibitor of the present invention can beformulated into, for example, a pharmaceutical composition such as atablet (including sugar-coated tablet, film-coated tablet), a powder, agranule, a capsule (including soft capsule), a liquid, an injection, asuppository, a sustained-release preparation (e.g., sustained-releasemicrocapsule), or an immediate-release preparation by, for example,mixing dihydroquercetin or a pharmaceutically acceptable salt thereofwith a pharmacologically acceptable carrier according to a known method,and safely administered orally or parenterally (e.g., topical, rectal,intravenous administration etc.). The injection can be used forintravenous, intramuscular, subcutaneous or intraorgan administration,or can be directly administered to the lesion. The pharmacologicallyacceptable carrier is similar to those described for the above-mentioned(I).

These preparations can be produced by a known method generally used forthe preparation steps.

The content of dihydroquercetin or a pharmaceutically acceptable saltthereof in the medicament and the inhibitor of the present invention canbe appropriately selected according to the form of preparation and thelike.

For example, the content of dihydroquercetin or a pharmaceuticallyacceptable salt thereof is generally about 0.01 to about 99.99 wt %,preferably about 0.1 to about 90 wt %, relative to the wholepreparation.

While the content of the additive such as carrier and the like in themedicament and the inhibitor of the present invention varies dependingon the form of the preparation, it is generally about 1 to about 99.99wt %, preferably about 10 to about 90 wt %, relative to the wholepreparation.

The medicament and the inhibitor of the present invention preferablycontain metformin or a pharmaceutically acceptable salt thereof, sincemetformin or a pharmaceutically acceptable salt thereof produces asynergistic anti-malignant tumor effect. The “metformin or apharmaceutically acceptable salt thereof” is similar to those describedfor the above-mentioned (I).

The content of metformin or a pharmaceutically acceptable salt thereofis generally about 0.5 to about 100 wt %, preferably about 2.5 to about50 wt %, more preferably about 2.5 to about 10 wt %, per 1 part byweight of dihydroquercetin.

The medicament and the inhibitor of the present invention can be used incombination with gemcitabine.

The dose of gemcitabine can be determined according to the dose ofcommercially available gemcitabine preparations.

The dose of the medicament and the inhibitor of the present inventioncan be appropriately selected according to the use, age and sex of thepatient, level of the disease and the like. The dose for an adult (bodyweight 60 kg) per day is generally about 40-1200 mg, preferably about50-900 mg, more preferably about 100-1050 mg, of dihydroquercetin or apharmaceutically acceptable salt thereof. The dose is administered inone to several portions per day.

EXAMPLES

The present invention is explained in detail in the following byreferring to Examples, which are not to be construed as limitative.

[Example 1] the Suppressive Effect on Proliferation-Related SignalPathway by Combined Use of Metformin and Dihydroquercetin in a HumanLung Cancer Cell Line NCI-H1299 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Metformin was dissolved in RPMI-1640 medium containing 10% inactivated(56° C., 30 min treatment) fetal bovine serum (hereinafter to beabbreviated as 10% FBS-RPMI1640 medium) (NACALAI TESQUE, INC.) toprepare 200 mmol/L metformin (final concentration 20 mmol/L).Dihydroquercetin was dissolved in ethanol, and diluted with medium toprepare 5 mmol/L dihydroquercetin (final concentration 0.5 mmol/L).

3) Cell

The human lung cancer-derived cell line NCI-H1299 was obtained fromAmerican Type Culture Collection (ATCC) (catalog no. CRL-5803, CancerRes. 1992; 52 (9 Suppl): 2732s-2736s). The cells were cultured in 10%FBS-RPMI1640 medium under the conditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, test substances were added andthe cells were cultured for 6 hr before harvesting them. The amounts ofrelative phospho-4E-BP1 protein contained in the prepared whole celllysates were measured and evaluated. Each group (N-3) was treated asfollows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 20 mmol/L(3) Dihydroquercetin single-treated group: dihydroquercetin 0.5 mmol/L(4) Combined-treated group: metformin 20 mmol/L+dihydroquercetin 0.5mmol/L

The passage-cultured NCI-H1299 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 1×10⁶ cells/mL. Thecells were seeded on 12-well multiwell culture plate (IWAKI) at 0.5 mLper well, and cultured overnight under the conditions of 37° C., 5% CO₂.On the following day, medium containing metformin or dihydroquercetin orboth metformin and dihydroquercetin, at the given concentration, wasadded into each well. After incubation for 6 hr, the cells in each wellwere washed twice with Dulbecco's phosphate buffered saline (D-PBS), andwhole cell lysates were prepared using cell lysis buffer (Cell SignalingTechnology, Inc.). Upon centrifugation, the supernatants were sampled,and the amounts of relative phospho-4E-BP1 (Thr37/Thr46) protein in thesamples were quantified using ELISA kits (Cell Signaling Technology,Inc., catalog No. 7854). The concentrations of the total protein in thecell extract were measured using BCA protein assay (Pierce), and therelative phospho-4E-BP1 protein amount of each well was normalized withthe total protein amount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of the combined use on relative phospho-4E-BP1protein amount, the Dunnett test was performed to compare thecombined-treated group with each single agent-treated group. When asignificant difference was observed in the combined-treated groupagainst each single agent treated group, two-way ANOVA (two-tailed)(factors: group and concentration) was performed to evaluate thesynergistic effect between the metformin-treated groups (groups 1 and 2)and the dihydroquercetin-treated groups (groups 3 and 4). The analysiswas performed using SAS Software Release 9.3 (SAS Institute Japan). Thedifferences were considered statistically significant when the p valuewas less than 0.05.

6) Results

The combined use of metformin and dihydroquercetin suppressed therelative phospho-4E-BP1 protein amount significantly stronger than eachsingle compound group (FIG. 1).

7) Conclusion

In NCI-H1299 cells, a synergistic suppressive effect onproliferation-related signal pathway by the combined use of metforminand dihydroquercetin was observed. This indicated the usefulness of acombination of metformin and dihydroquercetin in cancer chemotherapy.

[Example 2] the Suppressive Effect on Cancer Cell Proliferation-RelatedSignal Pathway by Combined Use of Metformin and Dihydroquercetin in aHuman Pancreatic Cancer Cell Line AsPC-1 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 200 mmol/Lmetformin (final concentration 20 mmol/L). Dihydroquercetin wasdissolved in ethanol, and diluted with medium to prepare 2 mmol/Ldihydroquercetin (final concentration 0.2 mmol/L).

3) Cell

Human pancreatic cancer-derived cell line AsPC-1 was obtained fromDainippon Pharmaceutical Co., Ltd. (now DS PHARMA BIOMEDICAL CO., LTD.,In Vitro. 1982; 18: 24-34). The cells were cultured in a 10%FBS-RPMI1640 medium under the conditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, test substances were added andthe cells were cultured for 6 hr before harvesting them. The amounts ofrelative phospho-4E-BP1 protein contained in the prepared whole celllysates were measured and evaluated. Each group (N=3) was treated asfollows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 20 mmol/L(3) Dihydroquercetin single-treated group: dihydroquercetin 0.2 mmol/L(4) Combined-treated group: metformin 20 mmol/L+dihydroquercetin 0.2mmol/L

The passage-cultured AsPc-1 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 1×10⁶ cells/mL. Thecells were seeded on 12-well multiwell culture plate at 0.5 mL per well,and cultured overnight under the conditions of 37° C., 5% CO₂. On thefollowing day, medium containing metformin or dihydroquercetin or bothmetformin and dihydroquercetin, at the given concentration, was addedinto each well. After incubation for 6 hr, the cells in each well werewashed twice with D-PBS, and whole cell lysates were prepared using celllysis buffer. Upon centrifugation, the supernatants were sampled, andthe amounts of relative phospho-4E-BP1 (Thr37/Thr46) protein in thesamples were quantified using ELISA kits in the same manner as inExample 1. The concentrations of the total protein in the whole celllysates were measured using BCA protein assay, and the relativephospho-4E-BP1 protein amount of each well was normalized with the totalprotein amount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of the combined use on relative phospho-4E-BP1protein amount, the Dunnett test was performed to compare thecombined-treated group with each single agent-treated group. When asignificant difference was observed in the combined-treated groupagainst each single agent-treated group, two-way ANOVA (two-tailed)(factors: group and concentration) was performed to evaluate thesynergistic effect between the metformin-treated groups (groups 1 and 2)and the dihydroquercetin-treated groups (groups 3 and 4). The analysiswas performed using SAS Software Release 9.3 (SAS Institute Japan). Thedifferences were considered statistically significant when the p valuewas less than 0.05.

6) Results

The combined use of metformin and dihydroquercetin suppressed therelative phospho-4E-BP1 protein amount significantly stronger than eachsingle compound group (FIG. 2).

7) Conclusion

In AsPC-1 cells, a synergistic suppressive effect onproliferation-related signal pathway by the combined use of metforminand dihydroquercetin was observed. This indicated the usefulness of acombination of metformin and dihydroquercetin in cancer chemotherapy.

[Example 3] the Suppressive Effect of Dihydroquercetin onMetformin-Induced Lactate Production in a Human Lung Fibroblast CellLine WI-38 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 200 mmol/Lmetformin (final concentration 20 mmol/L). Dihydroquercetin wasdissolved in ethanol, and diluted with medium to prepare 10 mmol/Ldihydroquercetin (final concentration 1 mmol/L).

3) Cell

Human lung fibroblast-derived cell line WI-38 was obtained from ATCC(catalog no. CCL-75, Exp. Cell. Res., 25: 585-621, 1961). The cells werecultured in 10% FBS-RPMI1640 medium under the conditions of 37° C., 5%CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, dihydroquercetin was added andthe cells were cultured for 1 hr. Then, metformin was added and thecells were cultured for 6 hr. After the culture, the conditioned mediumwas sampled, and the concentration of lactate contained in the mediumwas measured and evaluated. Each group (N=3) was treated as follows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 20 mmol/L(3) Dihydroquercetin and Metformin combined-treated group: metformin 20mmol/L+dihydroquercetin 1 mmol/L

The passage-cultured WI-38 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 2.5×10⁵ cells/mL. Thecells were seeded on 24-well multiwell culture plate (IWAKI) at 0.5 mLper well, and cultured overnight under the conditions of 37° C., 5% CO₂.Medium containing the given concentration of dihydroquercetin was addedto each well and the cells were cultured for 1 hr. Then, mediumcontaining the given concentration of metformin was added and the cellswere further cultured for 6 hr. After the culture, the culturesupernatant from each well was sampled and passed through a filter formolecular weight cut-off 10 kDa to obtain the filtrate. Using theLactate Assay Kit II (BioVision) catalog no. K627-100, lactateconcentration was measured. The cells after sampling of the culturesupernatant were washed twice with D-PBS, and cell lysis buffer wasadded to prepare whole cell lysates. The concentrations of the proteinin the whole cell lysates were measured by BCA protein assay, and thelactate concentration of each well was normalized with the proteinconcentration.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of dihydroquercetin on metformin-inducedproduced lactate content, a Student's t-test was performed for theconcentration of lactate in the medium between the metformin singletreated group and the combined-treated group. When the value in thecombined-treated group is significantly lower compared with themetformin single treated-group, dihydroquercetin is defined as having asuppressive effect on the content. The analysis was performed using SASsoftware Release 9.3 (SAS Institute Japan). The differences wereconsidered statistically significant when the p value was less than0.05.

6) Results

Metformin induced production of lactate in WI-38 cells. Dihydroquercetinsignificantly suppressed the lactate production induced by metformin(FIG. 3).

7) Conclusion

In WI-38 cells, a suppressive effect of dihydroquercetin onmetformin-induced lactate production was observed. This indicated aclinical usefulness of dihydroquercetin in reducing the risk of lacticacidosis caused by metformin.

[Example 4] the Suppressive Effect on Cancer Cell Proliferation-RelatedSignal Pathway by Combined Use of Metformin and Dihydroquercetin in aHuman Liver Cancer Cell Line HuH-7 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of test substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 50 mmol/Lmetformin (final concentration 5 mmol/L). Dihydroquercetin was dissolvedin ethanol, and diluted with medium to prepare 2 mmol/L dihydroquercetin(final concentration 0.2 mmol/L).

3) Cell

Human liver cancer-derived cell line HuH-7 was obtained from HealthScience Research Resources Bank (Cancer Res. 1982; 42 (9): 3858-63). Thecells were cultured in 10% FBS-RPMI1640 medium under the conditions of37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, test substances were added andthe cells were cultured for 6 hr before harvesting them. The amounts ofrelative phospho-4E-BP1 protein contained in the prepared whole celllysates were measured and evaluated. Each group (N=3) was treated asfollows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 5 mmol/L(3) Dihydroquercetin single-treated group: dihydroquercetin 0.2 mmol/L(4) Combined-treated group: metformin 5 mmol/L+dihydroquercetin 0.2mmol/L

The passage-cultured HuH-7 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 2.5×10⁵ cells/mL. Thecells were seeded on 12-well multiwell culture plate at 1 mL per well,and cultured overnight under the conditions of 37° C., 5% CO₂. On thefollowing day, medium containing metformin or dihydroquercetin or bothmetformin and dihydroquercetin, at the given concentration, was addedinto each well. After incubation for 6 hr, the cells in each well werewashed twice with D-PBS, and whole cell lysates were prepared using celllysis buffer. Upon centrifugation, the supernatants were sampled, andthe amounts of relative phospho-4E-BP1 (Thr37/Thr46) protein in thesamples were quantified using ELISA kits in the same manner as inExample 1. The concentrations of the total protein in the whole celllysates were measured using BCA protein assay, and the relativephospho-4E-BP1 protein amount of each well was normalized with the totalprotein amount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of the combined use on the relativephospho-4E-BP1 protein amount, the Dunnett test was performed to comparethe combined-treated group with each single agent treated group. When asignificant difference was observed in the combined-treated groupagainst each single agent-treated group, two-way ANOVA(two-tailed)(factors: group and concentration) was performed to evaluatethe synergistic effect between the metformin-treated groups (groups 1and 2) and the dihydroquercetin-treated groups (groups 3 and 4). Theanalysis was performed using SAS Software Release 9.3 (SAS InstituteJapan). The differences were considered statistically significant whenthe p value was less than 0.05.

6) Results

The results are shown in Table 1.

The combined use of metformin and dihydroquercetin suppressed therelative phospho-4E-BP1 protein amount significantly stronger than eachsingle compound group.

TABLE 1 Relative phospho-4E-BP1 protein amount MEAN SD Control 84.1 2.1Metformin 61.7 3.5 Dihydroquercetin 59.7 1.6 Metformin + 28.2 2.6Dihydroquercetin

7) Conclusion

In HuH-7 cells, a synergistic suppressive effect of cancer cellproliferation-related signal pathway by the combined use of metforminand dihydroquercetin was observed. This indicated the usefulness of acombination of metformin and dihydroquercetin in cancer chemotherapy.

[Example 5] the Suppressive Effect on Cancer Cell Proliferation-RelatedSignal Pathway by Combined Use of Metformin and Dihydroquercetin in aHuman Breast Cancer Cell Line MDA-MB-231 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 50 mmol/Lmetformin (final concentration 5 mmol/L). Dihydroquercetin was dissolvedin ethanol, and diluted with medium to prepare 2 mmol/L dihydroquercetin(final concentration 0.2 mmol/L).

3) Cell

Human breast cancer-derived cell line MDA-MB-231 was obtained from ATCC(J Natl Cancer Inst. 1974; 53 (3): 661-74). The cells were cultured in10% FBS-RPMI1640 medium under the conditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, test substances were added andthe cells were cultured for 6 hr before harvesting them. The amounts ofrelative phospho-4E-BP1 protein contained in the prepared whole celllysates were measured and evaluated. Each group (N=3) was treated asfollows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 5 mmol/L(3) Dihydroquercetin single-treated group: dihydroquercetin 0.2 mmol/L(4) Combined-treated group: metformin 5 mmol/L+dihydroquercetin 0.2mmol/L

The passage-cultured MDA-MB-231 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometor and adjusted to 2.5×10⁵ cells/mL. Thecells were seeded on 12-well multiwell culture plate at 1 mL per well,and cultured overnight under the conditions of 37° C., 5% CO₂. On thefollowing day, medium containing metformin or dihydroquercetin or bothmetformin and dihydroquercetin, at the given concentration, was addedinto each well. After incubation for 6 hr, the cells in each well werewashed twice with D-PBS, and whole cell lysates were prepared using celllysis buffer. Upon centrifugation, the supernatants were sampled, andthe amounts of relative phospho-4E-BP1 (Thr37/Thr46) protein in thesamples were quantified using ELISA kits in the same manner as inExample 1. The concentrations of total protein in the whole cell lysateswere measured using BCA protein assay, and the relative phospho-4E-BP1protein amount of each well was normalized with the total proteinamount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of the combined use on the relativephospho-4E-BP1 protein amount, the Dunnett test was performed to comparethe combined-treated group with each single agent treated group. When asignificant difference was observed in the combined-treated groupagainst each single agent-treated group, two-way ANOVA (two-tailed)(factors: group and concentration) was performed to evaluate thesynergistic effect between the metformin-treated groups (groups 1 and 2)and the dihydroquercetin-treated groups (groups 3 and 4). The analysiswas performed using SAS Software Release 9.3 (SAS Institute Japan). Thedifferences were considered statistically significant when the p valuewas less than 0.05.

6) Results

The results are shown in Table 2.

The combined-use of metformin and dihydroquercetin suppressed therelative phospho-4E-BP1 protein amount significantly stronger than eachsingle compound group.

TABLE 2 Relative phospho-4E-BP1 protein amount MEAN SD Control 33.3 3.1Metformin 37.1 3.1 Dihydroquercetin 25.9 0.6 Metformin + 19.8 1.4Dihydroquercetin

7) Conclusion

In MDA-MB-231 cells, a synergistic suppressive effect onproliferation-related signal pathway by the combined use of metforminand dihydroquercetin was observed. This indicated the usefulness of acombination of metformin and dihydroquercetin in cancer chemotherapy.

[Example 6] the Suppressive Effect on Cancer Cell Proliferation-RelatedSignal Pathway by Combined Use of Metformin and Dihydroquercetin in aHuman Prostate Cancer Cell Line 22Rv1 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 100 mmol/Lmetformin (final concentration 10 mmol/L). Dihydroquercetin wasdissolved in ethanol, and diluted with medium to prepare 2 mmol/Ldihydroquercetin (final concentration 0.2 mmol/L).

3) Cell

Human prostate cancer-derived cell line 22Rv1 was obtained from ATCC (InVitro Cell Dev Biol Anim. 1999; 35 (7): 403-9). The cells were culturedin 10% FBS-RPMI1640 medium under the conditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, test substances were added andthe cells were cultured for 6 hr before harvesting them. The amounts ofrelative phospho-4E-BP1 protein contained in the prepared whole celllysates were measured and evaluated. Each group (N=3) was treated asfollows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 10 mmol/L(3) Dihydroquercetin single-treated group: dihydroquercetin 0.2 mmol/L(4) Combined-treated group: metformin 10 mmol/L+dihydroquercetin 0.2mmol/L

The passage-cultured 22Rv1 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 2.5×10⁵ cells/mL. Thecells were seeded on 12-well multiwell culture plate at 1 mL per well,and cultured overnight under the conditions of 37° C., 5% CO₂. On thefollowing day, medium containing metformin or dihydroquercetin or bothmetformin and dihydroquercetin, at the given concentration, was addedinto each well. After incubation for 6 hr, the cells in each well werewashed twice with D-PBS, and whole cell lysates were prepared using celllysis buffer. Upon centrifugation, the supernatants were sampled, andthe amounts of relative phospho-4E-BP1 (Thr37/Thr46) protein in thesamples were quantified using ELISA kits in the same manner as inExample 1. The concentrations of total protein in the whole cell lysateswere measured using BCA-protein assay, and the relative phospho-4E-BP1protein amount of each well was normalized with the total proteinamount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of the combined use on the relativephospho-4E-BP1 protein amount, the Dunnett test was performed to comparethe combined-treated group with each single agent treated group. When asignificant difference was observed in the combined-treated groupagainst each single agent-treated group, two-way ANOVA (two-tailed)(factors: group and concentration) was performed to evaluate thesynergistic effect between the metformin-treated groups (groups 1 and 2)and the dihydroquercetin-treated groups (groups 3 and 4). The analysiswas performed using SAS Software Release 9.3 (SAS Institute Japan). Thedifferences were considered statistically significant when the p valuewas less than 0.05.

6) Results

The results are shown in Table 3.

The combined use of metformin and dihydroquercetin suppressed therelative phospho-4E-BP1 protein amount significantly stronger than eachsingle compound group.

TABLE 3 Relative phospho-4E-BP1 protein amount MEAN SD Control 51.0 6.7Metformin 57.3 2.2 Dihydroquercetin 53.1 4.5 Metformin + 36.2 1.5Dihydroquercetin

7) Conclusion

In 22Rv1 cells, a synergistic suppressive effect onproliferation-related signal pathway by the combined use of metforminand dihydroquercetin was observed. This indicated the usefulness of acombination of metformin and dihydroquercetin in cancer chemotherapy.

[Example 7] the Suppressive Effect on Cancer Cell Proliferation-RelatedSignal Pathway by Combined Use of Metformin and Dihydroquercetin in aHuman Bile Duct Cancer Cell Line HuH-28 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 200 mmol/Lmetformin (final concentration 20 mmol/L). Dihydroquercetin wasdissolved in ethanol, and diluted with medium to prepare 2 mmol/Ldihydroquercetin (final concentration 0.2 mmol/L).

3) Cell

Human bile duct cancer-derived cell line HuH-28 was obtained from HealthScience Research Resources Bank (Res Exp Med (Berl). 1988; 188(5):367-75). The cells were cultured in 10% FBS-RPMI1640 medium under theconditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, test substances were added andthe cells were cultured for 6 hr before harvesting them. The amounts ofrelative phospho-4E-BP1 protein contained in the prepared whole celllysates were measured and evaluated. Each group (N=3) was treated asfollows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 20 mmol/L(3) dihydroquercetin single-treated group: dihydroquercetin 0.2 mmol/L(4) combined-treated group: metformin 20 mmol/L+dihydroquercetin 0.2mmol/L

The passage-cultured HuH-28 cells were dissociated with trypsin, andsuspended in the medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 2.5×10⁵ cells/mL. Thecells were seeded on 12-well multiwell culture plate at 1 mL per well,and cultured overnight under the conditions of 37° C., 5% CO₂. On thefollowing day, medium containing metformin or dihydroquercetin or bothmetformin and dihydroquercetin, at the given concentration, was addedinto each well. After incubation for 6 hr, the cells in each well werewashed twice with D-PBS, and whole cell lysates were prepared using acell lysis buffer. Upon centrifugation, the supernatants were sampled,and the amounts of relative phospho-4E-BP1 (Thr37/Thr46) protein in thesamples were quantified using ELISA kits in the same manner as inExample 1. The total protein concentrations in the whole cell lysateswere measured using BCA protein assay, and the relative phospho-4E-BP1protein amount of each well was normalized with the total proteinamount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of the combined use on the relativephospho-4E-BP1 protein amount, the Dunnett test was performed to comparethe combined-treated group with each single agent treated group. When asignificant difference was observed in the combined-treated groupagainst each single agent-treated group, two-way ANOVA (two-tailed)(factors: group and concentration) was performed to evaluate thesynergistic effect between the metformin-treated groups (groups 1 and 2)and the dihydroquercetin-treated groups (groups 3 and 4). The analysiswas performed using SAS Software Release 9.3 (SAS Institute Japan). Thedifferences were considered statistically significant when the p valuewas less than 0.05.

6) Results

The results are shown in Table 4.

The combined use of metformin and dihydroquercetin suppressed therelative phospho-4E-BP1 protein amount significantly stronger than eachsingle compound group.

TABLE 4 Relative phospho-4E-BP1 protein amount MEAN SD Control 58.8 2.8Metformin 55.2 1.5 Dihydroquercetin 54.2 0.2 Metformin + 36.6 1.3Dihydroquercetin

7) Conclusion

In HuH-28 cells, a synergistic suppressive effect onproliferation-related signal pathway by the combined use of metforminand dihydroquercetin was observed. This indicated the usefulness of acombination of metformin and dihydroquercetin in cancer chemotherapy.

[Example 8] the Suppressive Effect on Cancer Cell Proliferation RelatedSignal Pathway by Combined Use of Metformin and Dihydroquercetin in aHuman Ovarian Cancer Cell Line Caov-3 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 200 mmol/Lmetformin (final concentration 20 mmol/L). Dihydroquercetin wasdissolved in ethanol, and diluted with medium to prepare 2 mmol/Ldihydroquercetin (final concentration 0.2 mmol/L).

3) Cell

Human ovarian cancer-derived cell line Caov-3 was obtained from ATCC(GYNECOL ONCOL. 1994; 53(1): 70-7). The cells were cultured in 10%FBS-RPMI1640 medium under the conditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, test substances were added andthe cells were cultured for 6 hr before harvesting them. The amounts ofrelative phospho-4E-BP1 protein contained in the prepared whole celllysates were measured and evaluated. Each group (N=3) was treated asfollows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 20 mmol/L(3) Dihydroquercetin single-treated group: dihydroquercetin 0.2 mmol/L(4) Combined-treated group: metformin 20 mmol/L+dihydroquercetin 0.2mmol/L

The passage-cultured Caov-3 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 2.5×10⁵ cells/mL. Thecells were seeded on 12-well multiwell culture plate at 1 mL per well,and cultured overnight under the conditions of 37° C., 5% CO₂. On thefollowing day, medium containing metformin or dihydroquercetin or bothmetformin and dihydroquercetin, at the given concentration, was addedinto each well. After incubation for 6 hr, the cells in each well werewashed twice with D-PBS, and whole cell lysates were prepared using celllysis buffer. Upon centrifugation, the supernatants were sampled, andthe amounts of relative phospho-4E-BP1 (Thr37/Thr46) protein in thesamples were quantified using ELISA kits in the same manner as inExample 1. The total protein concentrations in the whole cell lysateswere measured using BCA protein assay, and the relative phospho-4E-BP1protein amount of each well was normalized with the total proteinamount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of the combined use on the relativephospho-4E-BP1 protein amount, the Dunnett test was performed to comparethe combined-treated group with each single agent treated group. When asignificant difference was observed in the combined-treated groupagainst each single agent-treated group, two-way ANOVA (two-tailed)(factors: group and concentration) was performed to evaluate thesynergistic effect between the metformin-treated groups (groups 1 and 2)and the dihydroquercetin-treated groups (groups 3 and 4). The analysiswas performed using SAS Software Release 9.3 (SAS Institute Japan). Thedifferences were considered statistically significant when the p valuewas less than 0.05.

6) Results

The results are shown in Table 5.

The combined use of metformin and dihydroquercetin suppressed therelative phospho-4E-BP1 protein amount significantly stronger than eachsingle compound group.

TABLE 5 Relative phospho-4E-BP1 protein amount MEAN SD Control 21.6 2.7Metformin 22.0 0.3 Dihydroquercetin 21.0 1.0 Metformin + 16.8 0.3Dihydroquercetin

7) Conclusion

In Caov-3 cells, a synergistic suppressive effect onproliferation-related signal pathway by the combined use of metforminand dihydroquercetin was observed. This indicated the usefulness of acombination of metformin and dihydroquercetin in cancer chemotherapy.

[Example 9] the Suppressive Effect on Cancer Cell Proliferation-RelatedSignal Pathway by Combined Use of Metformin and Dihydroquercetin in aHuman Pancreatic Cancer Cell Line AsPC-1 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 15, 30 and60 mmol/L metformin (final concentrations 1.5, 3 and 6 mmol/L).Dihydroquercetin was dissolved in ethanol, and diluted with medium toprepare 3 mmol/L dihydroquercetin (final concentration 0.3 mmol/L).

3) Cell

Human pancreatic cancer-derived cell line AsPC-1 was obtained from DSPHARMA BIOMEDICAL CO., LTD. The cells were cultured in 10% FBS-RPMI1640medium under the conditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

Each group (N=3) was treated as follows.

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Metformin single-treated group: metformin 1.5, 3 and 6 mmol/L(3) Dihydroquercetin-single treated group: dihydroquercetin 0.3 mmol/L(4) Combined-treated group: metformin 1.5, 3 and 6mmol/L+dihydroquercetin 0.3 mmol/L

The passage-cultured AsPC-1 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 2.5×10⁵ cells/mL. Thecells were seeded on 12-well multiwell culture plate at 1 mL per well,and cultured overnight under the conditions of 37° C., 5% CO₂. On thefollowing day, medium containing metformin or dihydroquercetin or bothmetformin and dihydroquercetin, at the given concentration, was addedinto each well. After incubation under the conditions of 37° C., 5% CO₂for 24 hr, the cells in each well were washed twice with D-PBS, andwhole cell lysates were prepared using cell lysis buffer. Uponcentrifugation, the supernatants were sampled, and the amounts ofrelative phospho-4E-BP1 (Thr37/Thr46) protein in the samples werequantified using ELISA kits in the same manner as in Example 1. Theconcentrations of total protein in the whole cell lysates were measuredusing BCA protein assay, and the relative phospho-4E-BP1 protein amountof each well was normalized with the total protein amount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of the combined use on relative phospho-4E-BP1protein amount, Dunnett test was performed to compare thecombined-treated group with each single agent-treated group. When asignificant difference was observed in the combined-treated groupagainst each single agent-treated group, two-way ANOVA(two-tailed)(factors: group and concentration) was performed to evaluatethe synergistic effect between the metformin-treated groups (groups 1and 2) and the dihydroquercetin-treated groups (groups 3 and 4). Theanalysis was performed using SAS Software Release 9.3 (SAS InstituteJapan). The differences were considered statistically significant whenthe p value was less than 0.05.

6) Results

The results are shown in Tables 6-8.

The combined use of metformin and dihydroquercetin suppressed therelative phospho-4E-BP1 protein amount significantly stronger than eachsingle compound group.

TABLE 6 metformin:dihydroquercetin = 1.5 mmol/L:0.3 mmol/L Relativephospho-4E-BP1 protein amount MEAN SD Control 107.3 9.3 Metformin 83.15.5 Dihydroquercetin 97.4 9.6 Metformin + 31.2 4.1 Dihydroquercetin

TABLE 7 metformin:dihydroquercetin = 3 mmol/L:0.3 mmol/L Relativephospho-4E-BP1 protein amount MEAN SD Control 33.5 10.1 Metformin 36.91.7 Dihydroquercetin 37.2 2.5 Metformin + 18.4 0.9 Dihydroquercetin

TABLE 8 metformin:dihydroquercetin = 6 mmol/L:0.3 mmol/L Relativephospho-4E-BP1 protein amount MEAN SD Control 107.3 9.3 Metformin 83.15.5 Dihydroquercetin 89.7 7.1 Metformin + 29.4 1.2 Dihydroquercetin

7) Conclusion

In AsPC-1 cells, a synergistic suppressive effect onproliferation-related signal pathway by the combined use of metforminand dihydroquercetin was observed. This indicated the usefulness of acombination of metformin and dihydroquercetin in cancer chemotherapy.

[Example 10] the Suppressive Effect of Dihydroquercetin onMetformin-Induced Lactate Production in a Human Lung Fibroblast CellLine WI-38 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Racemate of dihydroquercetin ((2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin)) was purchased from Bionet, andoptically-active dihydroquercetin ((2R,3R)-dihydroquercetin) waspurchased from SIGMA.

2) Preparation of Test Substances

Metformin was dissolved in 10% FBS-RPMI1640 medium to prepare 200 mmol/Lmetformin (final concentration 20 mmol/L). Dihydroquercetin wasdissolved in ethanol, and diluted with medium to prepare 10 mmol/Ldihydroquercetin (final concentration 1 mmol/L).

3) Cell

Human lung fibroblast-derived cell line WI-38 was obtained from ATCC(catalog no. CCL-75, Exp. Cell. Res., 25: 585-621, 1961). The cells werecultured in 10% FBS-RPMI1640 medium under the conditions of 37° C., 5%CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, dihydroquercetin was added andthe cells were cultured for 1 hr. Then, metformin was added and thecells were cultured for 6 hr. The conditioned medium was sampled, andthe concentration of the lactate contained in the medium was measuredand evaluated. Each group (N=3) was treated as follows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Dihydroquercetin single-treated group: dihydroquercetin 1 mmol/L(3) Metformin single-treated group: metformin 20 mmol/L(4) Combined-treated group: metformin 20 mmol/L+dihydroquercetin 1mmol/L

The passage-cultured WI-38 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 2.5×10⁵ cells/mL. Thecells were seeded on 24-well multiwell culture plate (IWAKI) at 0.5 mLper well, and cultured overnight under the conditions of 37° C., 5% CO₂.Thereto was added a medium containing a given concentration ofdihydroquercetin and the cells were cultured for 1 hr, a mediumcontaining a given concentration of metformin was added and the cellswere further cultured for 6 hr. After culture, the culture supernatantfrom each well was sampled and passed through a filter for molecularweight cut-off 10 kDa to obtain the filtrate. Using the Lactate AssayKit II (BioVision) catalog no. K627-100, lactate concentration wasmeasured. The cells after sampling of the culture supernatant werewashed twice with D-PBS, and cell lysis buffer was added to preparewhole cell lysates. The concentrations of the protein in the whole celllysates were measured using BCA protein assay, and the lactateconcentration of each well was normalized with the proteinconcentration.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of dihydroquercetin on the metformin-inducedproduced lactate content, the Student's t-test was performed for theconcentration of lactate in the medium between the control group and thedihydroquercetin single agent treated group, and between the controlgroup and the metformin single agent treated group. In addition, toevaluate an influence of the combination of dihydroquercetin andmetformin for normalized produced lactate content, a two-way ANOVA(two-tailed) (factors: group and concentration) was performed betweenthe dihydroquercetin single agent treated group (groups 1 and 2) and themetformin treated group (groups 3 and 4). When normalized producedlactate content in the dihydroquercetin single agent treated group issignificant compared to that in the control group, the agent was judgedto have a suppressive action on lactate production. When normalizedproduced lactate content in the metformin single agent treated group issignificant compared to that in the control group, the agent was judgedto have an inducing action on lactate production. When the difference innormalized produced lactate content between combined-treated group andmetformin single agent treated group was significantly larger than thatbetween the control group and dihydroquercetin single agent treatedgroup, dihydroquercetin was defined as having a suppressive effect onthe content. The analysis was performed using SAS software Release 9.3(SAS Institute Japan). The differences were considered statisticallysignificant when the p value was less than 0.05.

6) Results

Both the racemate and optically-active dihydroquercetin suppressedlactate production in WI-38 as compared with the control group.Metformin induced lactate production in the cells and this wassignificantly suppressed by both the racemate and optically-activedihydroquercetin (Racemate; FIG. 4, Optically-active; FIG. 5).

7) Conclusion

In WI-38 cells, a suppressive effect of dihydroquercetin onmetformin-induced lactate production by the racemate andoptically-active dihydroquercetin was observed. This indicated aclinical usefulness of dihydroquercetin in reducing the risk of lacticacidosis caused by metformin administration.

[Example 11] the Suppressive Effect on Cancer Cell Proliferation-RelatedSignal Pathway by Dihydroquercetin Single Agent in a Human PancreaticCancer Cell Line AsPC-1 (In Vitro) 1) Test Substances

Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet.

2) Preparation of Test Substances

Dihydroquercetin was dissolved in ethanol, and diluted with medium toprepare 5 and 10 mmol/L dihydroquercetin (final concentration 0.5 and 1mmol/L).

3) Cell

The human pancreatic cancer-derived cell line AsPC-1 was obtained fromDainippon Pharmaceutical Co., Ltd. (now DS PHARMA BIOMEDICAL CO., LTD.,In Vitro. 1982; 18: 24-34). The cells were cultured in 10% FBS-RPMI1640medium under the conditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

On the following day after cell seeding, test substances were added andthe cells were cultured for 6 hr before harvesting them. The amounts ofrelative phospho-4E-BP1 protein contained in the prepared whole celllysates were measured and evaluated. Each group (N=3) was treated asfollows:

(1) Control (vehicle control) group: 1% ethanol containing medium(2) Dihydroquercetin single-treated group: dihydroquercetin 0.5 mmol/L(3) Dihydroquercetin single-treated group: dihydroquercetin 1 mmol/L

The passage-cultured AsPC-1 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 1×10⁶ cells/mL. Thecells were seeded on 12-well multiwell culture plate at 0.5 mL per well,and cultured overnight under the conditions of 37° C., 5% CO₂. On thefollowing day, medium containing dihydroquercetin, at the givenconcentration, was added into each well. After incubation for 6 hr, thecells in each well were washed twice with D-PBS, and whole cell lysateswere prepared using cell lysis buffer. Upon centrifugation, thesupernatant was sampled, and the amounts of relative phospho-4E-BP1(Thr37/Thr46) protein in the samples were quantified using ELISA kits inthe same manner as in Example 1. The concentrations of the total proteinin the whole cell lysates were measured using BCA protein assay, and therelative phospho-4E-BP1 protein amount of each well was normalized withthe total protein amount.

5) Statistical Analysis

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the dose dependency of dihydroquercetin on the relativephospho-4E-BP1 protein amount, the linear regression analysis wasperformed. As a result, dose dependency was observed. Then, theWilliams' test (one-tailed) was performed between the control group andthe single agent treated group. As a result, a significant suppressiveaction of dihydroquercetin at not less than 0.5 mmol/L on relativephospho-4E-BP1 protein amount was observed. When relative phospho-4E-BP1protein amount in the dihydroquercetin single agent-treated group issignificant compared to that in the control group, the agent was judgedto have a suppressive action on the amount. The analysis was performedusing SAS software Release 9.3 (SAS Institute Japan). The differenceswere considered statistically significant when the p value was less than0.05.

6) Results

Dihydroquercetin showed a dose-dependent suppressive effect on therelative phospho-4E-BP1 protein amount (FIG. 6).

7) Conclusion

In AsPC-1 cells, a dose-dependent suppressive effect onproliferation-related signal pathway by the dihydroquercetin singleagent was observed. This indicated the usefulness of dihydroquercetin incancer chemotherapy.

[Example 12] the Suppressive Effect on Surface Marker Expression ofCancer Stem Cells by Combined Use of Metformin and Dihydroquercetin in aHuman Pancreatic Cancer Cell Line AsPC-1 (In Vitro) 1) Test Substances

Metformin was purchased from Wako Pure Chemical Industries, Ltd.Dihydroquercetin (racemate of (2R,3R)-dihydroquercetin and(2S,3S)-dihydroquercetin) was purchased from Bionet. Gemcitabine waspurchased from Toronto Research Chemicals Inc.

2) Preparation of Test Substances

Metformin was dissolved in distilled water, and diluted with 10%FBS-RPMI1640 medium to prepare 15 mmol/L metformin. Dihydroquercetin wasdissolved in ethanol, and diluted with medium to prepare 0.3 mmol/Ldihydroquercetin. Gemcitabine was dissolved in dimethyl sulfoxide(DMSO), and diluted with medium to prepare 100 nmol/L gemcitabine.

3) Cell

Human pancreatic cancer-derived cell line AsPC-1 was obtained from DSPHARMA BIOMEDICAL CO., LTD. The cells were cultured in 10% FBS-RPMI1640medium under the conditions of 37° C., 5% CO₂.

4) Addition of Test Substances and Measurement

Each group (N=10) was treated as follows:

(1) Control (vehicle control) group: 1.5% distilled water, 0.3% ethanoland 0.1% DMSO containing medium(2) Metformin single-treated group: metformin 15 mmol/L(3) Dihydroquercetin single-treated group: dihydroquercetin 0.3 mmol/L(4) Metformin-dihydroquercetin combined-treated group: metformin 15mmol/L+dihydroquercetin 0.3 mmol/L(5) Gemcitabine single-treated group: gemcitabine 100 nmol/L(6) Metformin⋅dihydroquercetin⋅gemcitabine combined-treated group:metformin 15 mmol/L+dihydroquercetin 0.3 mmol/L+gemcitabine 100 nmol/L

The passage-cultured AsPC-1 cells were dissociated with trypsin, andsuspended in fresh medium. The cell density of the cell suspension wasdetermined using a hematocytometer and adjusted to 2×10⁴ cells/mL. Thecells were seeded on 6-well multiwell culture plate (IWAKI) at 3 mL perwell, and cultured so overnight under the conditions of 37° C., 5% CO₂.On the following day, the medium was changed with a medium containingeach test substance at the given concentration, and the cells werecultured for 72 hr. The end of the culture, the cells were dissociatedwith trypsin, and suspended in fresh medium. After washing with FACS(fluorescence activated cell sorting) buffer, the cells were stainedwith FITC-labeled anti-human CD44 antibody and APC-labeled anti-humanCD24 antibody, and treated at 4° C. for 30 min. Thereafter, the cellswere washed with the FACS buffer, passed through a 40 micron meshfilter, and the proportion of CD44 and CD24 double positive cells wasdetermined using the flow cytometer (Nippon Becton Dickinson andCompany, Ltd.).

5) Statistical analyses

The results are shown in mean value (MEAN)±standard deviation (SD).

To evaluate the influence of metformin and dihydroquercetinsingle-treated group and a combined use group on the proportion of CD44and CD24 double positive cells, unpaired two-way ANOVA was performed tocompare the control group with each substance single-treated group andcombined-treated group. To evaluate the influence of the gemcitabinesingle-treated group on the proportion of CD44 and CD24 double positivecells, student's t-test was performed to compare the control group withthe gemcitabine single-treated group. In addition, to evaluate theinfluence of the gemcitabine, metformin and dihydroquercetincombined-treated group on the proportion of CD44 and CD24 doublepositive cells against the gemcitabine single-treated group, Student'st-test was performed to compare the gemcitabine single-treated group andthe gemcitabine, metformin and dihydroquercetin combined-treated group.The analysis was performed using SAS software Release 9.3 (SAS InstituteJapan). The differences were considered statistically significant whenthe p value was less than 0.05.

6) Results

The dihydroquercetin single-treated group and the metformin anddihydroquercetin combined-treated group significantly decreased theproportion of CD44 and CD24 double positive cells in AsPC-1 cells, ascompared with the control group. Gemcitabine single-treated groupsignificantly increased the proportion of CD44 and CD24 double positivecells in AsPC-1 cells, as compared with the control group. The increasedproportion of CD44 and CD24 double positive cells by gemcitabine singleaddition was significantly suppressed by the supplemental addition ofmetformin and dihydroquercetin (FIG. 7).

7) Conclusion

After evaluating the proportion of CD24 and CD44 double positive cells,determined as the cancer stem cells in pancreatic cancer, on pancreaticcancer cell line AsPC-1, the significant decrease were observed by thedihydroquercetin single addition and the combined addition of metforminand dihydroquercetin was observed. On the contrary, the significantincrease in the proportion of the double positive cells by gemcitabinewas observed. In addition, the above-mentioned increase of the CD24 andCD44 double positive cells by gemcitabine single addition wassignificantly decreased by the combined addition of metformin anddihydroquercetin. These results suggest that clinical problems, theincrease of recurrence risk depending on enrichment of cancer stem cellsassociated with gemcitabine treatment, may be solved by the combined useof gemcitabine and dihydroquercetin or the combined use of gemcitabine,metformin and dihydroquercetin.

INDUSTRIAL APPLICABILITY

According to the present invention, a medicament having a synergisticanti-malignant tumor effect and reduced side effects, which is useful asan anti-malignant tumor drug, can be provided by combining metformin ora pharmaceutically acceptable salt thereof and dihydroquercetin or apharmaceutically acceptable salt thereof.

This application is based on a patent application No. 2013-110278 filedin Japan, the contents of which are incorporated in full herein.

1-27. (canceled)
 28. A method of reducing the side effects of metforminor a pharmaceutically acceptable salt thereof, comprising administeringdihydroquercetin or a pharmaceutically acceptable salt thereof to apatient with a malignant tumor.
 29. A method of reducing the sideeffects of metformin or a pharmaceutically acceptable salt thereof,comprising administering dihydroquercetin or a pharmaceuticallyacceptable salt thereof, in combination with metformin or apharmaceutically acceptable salt thereof, to a patient with a malignanttumor.